Field of the Invention
The present invention in the field of molecular biology and medicine relates to the use of microbial-type rhodopsins, such as the light-gated cation-selective membrane channel, channelrhodopsin-2 (Chop2) to convert inner retinal neurons to photosensitive cells in photoreceptor-degenerated retina, thereby restoring visual perception and various aspects of vision.
Description of the Background Art
Vision normally begins when rods and cones, also called photoreceptors, convert light signals to electrical signals that are then relayed through second- and third-order retinal neurons and the optic nerve to the lateral geniculate nucleus and, then to the visual cortex where visual images are formed (Baylor, D, 1996, Proc. Natl. Acad. Sci. USA 93:560-565; Wässle, H, 2004, Nat. Rev. Neurosci. 5:747-57). For a patient who is vision-impaired due to the loss of photoreceptors, visual perception may be induced by providing electrical stimulation at one of these downstream neuronal locations, depending on the nature of the particular impairment.
The severe loss of photoreceptor cells can be caused by congenital retinal degenerative diseases, such as retinitis pigmentosa (RP) (Sung, C H et al., 1991, Proc. Natl. Acad. Sci. USA 88:6481-85; Humphries, P et al., 1992, Science 256:804-8; Weleber, R G et al., in: S J Ryan, Ed, Retina, Mosby, St. Louis (1994), pp. 335-466), and can result in complete blindness. Age-related macular degeneration (AMD) is also a result of the degeneration and death of photoreceptor cells, which can cause severe visual impairment within the centrally located best visual area of the visual field.
Both rodents and humans go progressively blind because, as rods and cones are lost, there is little or no signal sent to the brain. Inherited retinal degenerations that cause partial or total blindness affect one in 3000 people worldwide. Patients afflicted with Usher's Syndrome develop progressive deafness in addition to retinal degeneration. There are currently no effective treatments or cures for these conditions.
Basic research on approaches for retinal degeneration has long been classified into two approaches: (1) treatments to preserve remaining photoreceptors in patients with retinal degenerative disease, and (2) methods to replace photoreceptors lost to retinal degeneration. Patients afflicted with retinal disease often group themselves into those seeking ways to slow the loss of their diminishing vision and those who are already legally blind (“no light perception”), having lost their photoreceptors because of an inherited eye disease or trauma.
For the first approach, neuroprotection with neurotrophic factors (LaVail, M M et al., 1992, Proc. Natl. Acad. Sci. USA 89:11249-53) and virus-vector-based delivery of wild-type genes for recessive null mutations (Acland, G M et al., 2001, Nat. Genet. 28:92-95) have come the furthest—to the point of a Phase I/II clinical trial (Hauswirth, W W, 2005, Retina 25, S60; Jacobson, S, Protocol #0410-677, World Wide Web URL: webconferences.com/nihoba/16_jun_2005.html) gaining approval in the U.S. for adeno-associated viral (AAV)-mediated gene replacement therapy for Leber's Congenital Amaurosis (LCA), a specific form of retinal degeneration.
Unfortunately, for patients in advanced stages of retinal degeneration, this approach is not applicable, and the photoreceptor cells must be replaced.
For replacement, one approach involves transplantation (replacement) of normal tissues or cells to the diseased retina. Another involves electrical-stimulation of remaining non-visual neurons via retinal implants in lieu of the lost photoreceptive cells (prosthetic substitution). However, both methods face many fundamental obstacles. For example, for successful transplantation, the implanted tissue or cells must integrate functionally within the host retina. The electrical-stimulation approaches are burdened with mechanistic and technical difficulties as well as problems related to lack of long-term biocompatibility of the implanted bionic devices. In summary, there exist no effective vision-restoring therapies for inherited blinding disease.
The present inventors' strategy as disclosed herein, requires a suitable molecular “light-sensor.” Previous studies reported the heterologous expression of Drosophila rhodopsin (Zemelman, B V et al., 2002, Neuron 33:15-22) and, more recently, melanopsin, the putative photopigment of the intrinsic photosensitive retinal ganglion cells (McIyan, Z. et al., 2005, Nature 433:741-5; Panda, S. et al., 2005, Science 307:600-604; Qiu, X. et al., 2005, Nature 433:745-9). These photopigments, however, are coupled to membrane channels via a G protein signaling cascade and use cis-isoforms of retinaldehyde as their chromophore. As a result, expression of multiple genes would be required to render photosensitivity. In addition, their light response kinetics is rather slow. Recent studies aimed to improve the temporal resolution described the engineering of a light-sensitive K+ channel (Banghart et al., 2004, Nat. Neurosci. 7:1381-6), though this required introduction of an exogenous “molecular tether” and use of UV light to unblock the channel. This engineered channel was proposed to be potentially useful for restoring light sensitivity in degenerate retinas, but its expression and function in retinal neurons remain unknown.
The present invention makes use of microbial-type rhodopsins similar to bacteriorhodopsin (Oesterhelt, D et al., 1973, Proc. Natl. Acad. Sci. USA 70:2853-7), whose conformation change is caused by reversible photoisomerization of their chromophore group, the all-trans isoform of retinaldehyde, and is directly coupled to ion movement through the membrane (Oesterhelt, D., 1998, Curr. Opin. Struct. Biol. 8:489-500). Two microbial-type opsins, channelopsin-1 and -2 (Chop1 and Chop2), have recently been cloned from Chlamydomonas reinhardtii (Nagel, G. et al., 2002, Science 296:2395-8; Sineshchekov, O A et al., 2002, Proc. Natl. Acad. Sci. USA 99:8689-94; Nagel, G. et al., 2003, Proc. Natl. Acad. Sci. USA 100, 13940-45) and shown to form directly light-gated membrane channels when expressed in Xenopus laevis oocytes or HEK293 cells in the presence of all-trans retinal. Chop2, a seven transmembrane domain protein, becomes photo-switchable when bound to the chromophore all-trans retinal. Chop2 is particularly attractive because its functional light-sensitive channel, channelrhodopsin-2 (Chop2 retinalidene abbreviated ChR2) with the attached chromophore is permeable to physiological cations. Unlike animal rhodopsins, which only bind the 11-cis conformation, Chop2 binds all-trans retinal isomers, obviating the need for the all-trans to 11-cis isomerization reaction supplied by the vertebrate visual cycle. However, the long-term compatibility of expressing ChR2 in native neurons in vivo in general and the properties of ChR2-mediated light responses in retinal neurons in particular remained unknown until the present invention.
The present strategy is feasible because histological studies, both in animal models of photoreceptor degeneration (Chang, B. et al., 2002, Vision Res. 42:517-25; Olshevskaya, E V et al., 2004, J. Neurosci. 24:6078-85) and in postmortem patient eyes with almost complete photoreceptor loss due to RP (Santos, A H et al., 1997, Arch. Ophthalmol. 115:511-15; Milam, A H et al., 1998, Prog. Retin. Eye Res. 17:175-205), reported the preservation of a significant number of inner retinal neurons.
Retinal gene therapy has been considered a possible therapeutic option for man. For example, U.S. Pat. No. 5,827,702 refers to methods for generating a genetically engineered ocular cell by contacting the cell with an exogenous nucleic acid under conditions in which the exogenous nucleic acid is taken up by the cell for expression. The exogenous nucleic acid is described as a retrovirus, an adenovirus, an adeno-associated virus or a plasmid. See, also, WO 00/15822 (Mar. 23, 2000) and WO 98/48097 (Oct. 29, 1998)
Efforts in such gene therapy have focused mainly on slowing down retinal degeneration in rodent models of primary photoreceptor diseases. Normal genes and mutation-specific ribozymes delivered to photoreceptors have prolonged the lifetime of these cells otherwise doomed for apoptotic cell death (Bennett, J., et al. 1996 Nat. Med. 2, 649-54; Bennett, J., et al. 1998, Gene Therapy 5, 1156-64; Kumar-Singh, R et al., 1998 Hum. Mol. Genet. 7, 1893-900; Lewin, A S et al. 1998, Nat. Med. 4, 967-71; Ali, R et al. 2000, Nat. Genet. 25, 306-10; Takahashi, M. et al., 1999, J Virol. 73, 7812-6; Lau, D., et al., 2000, Invest. Ophthalmol. Vis. Sci. 41, 3622-33; and LaVail, M M, et al. 2000, Proc Natl Acad Sci USA 97, 11488-93).
Retinal gene transfer of a reporter gene, green fluorescent protein (GFP), using a recombinant adeno-associated virus (rAAV) was demonstrated in normal primates (Bennett, J et al. 1999 Proc. Natl. Acad. Sci. USA 96, 9920-25). However, the restoration of vision in a blinding disease of animals, particularly in humans and other mammals, caused by genetic defects in retinal pigment epithelium (RPE) and/or photoreceptor cells has not been achieved. Jean Bennett and colleagues have described the rescue of photoreceptors using gene therapy in a model of rapid degeneration of photoreceptors using mutations of the RP65 gene and replacement therapy with the normal gene to replace or supplant the mutant gene. See, for example, US Patent Publication 2004/0022766 of Acland, Bennett and colleagues. This therapy showed some success in a naturally-occurring dog model of severe disease of retinal degenerations—the RPE65 mutant dog, which is analogous to human LCA.
Advantages of the present approach include the fact that it does not require introducing exogenous cells and tissues or physical devices, thus avoiding many obstacles encountered by existing approaches; the present invention is applicable for the reversal of vision loss or blindness caused by many retinal degenerative diseases. By expressing photosensitive membrane-channels or molecules in surviving retinal neurons of the diseased retina by viral based gene therapy method, the present invention can produce permanent treatment of the vision loss or blindness with high spatial and temporal resolution for the restored vision.
To the extent that any specific disclosure in the aforementioned publications or other publications may be considered to anticipate any generic aspect of the present invention, the disclosure of the present invention should be understood to include a proviso or provisos that exclude of disclaim any such species that were previously disclosed. The aspects of the present invention which are not anticipated by the disclosure of such publications are also unobvious from the disclosure of these publications, due at least in part to the unexpectedly superior results disclosed or alleged herein.